Background: In the past year, we have extended our research to reaffirm the results described above by repeating the experiments with MORAb-009 labeled with Zr-89 (a positron emitter; decay half-life of 3.3 days) which better matches with the biological half-life of mAbs than Cu-64 (decay half-life, 12.7 h) labeled mAb, thus making the Zr-89 labeled mAb more relevant for the diagnosis and the assessment of the therapeutic responses of cancer. As a part of our mechanistic studies on the tumor uptake and microdistribution of MORAb-009 (anti-mesothelin mAb amatuzimab), we compared the results of Zr-89-MORAb-009 studies with those of Zr-89-labeled B3, a monoclonal antibody directed against Lewis-Y antigen which is not shed from tumor surface. Objectives: To develop a method to radiolabel two mAbs (MORAb-009 and anti-Lewis-Y mAb B3) and investigate the effect of the injection dose and the tumor size on the tumor uptake and microdistribution of these two Zr-89-labeled mAbs in nude mice bearing A431/H9 which expresses both mesothelin and Lewis-Y antigen. Methods: The mAbs were radiolabeled with Zr-89 using desferrioxamine with an isothiocyanate linker as a chelating agent. The Zr-89 labeled mAbs were then purified with a PD-10 column eluted with 0.25 M ammonium acetate at pH 5.5. The radiolabeled mAbs with the radiochemical purity >95% and the immunoreactivity >70% were used for in vivo studies. The biodistribution (BD) was performed in groups of nude mice (n=4-5) with A431/H9 tumors (range, 191 265 cubic mm for MORAb-009 and 260 405 cubic mm for B3 study) one day after iv injection of Zr-89-MORAb-009 (3 microCi/2, 10 or 60 microg) or Zr-89-B3 (3 microCi/2, 15 or 60 microg). For the BD studies, the animals were euthanized at 24h post-injection (p.i.) by CO2 inhalation and exsanguinated by cardiac puncture before dissection. Blood and various organs were removed and weighed, and their decay corrected radioactivity counts were measured with a gamma-counter (Wallac, Inc., Perkin-Elmer, Inc., Boston, MA). For PET imaging studies, the longitudinal 15 min static PET scans were performed on athymic mice (n=5) using a Siemens Inveon micro PET scanner (Siemens Preclinical Solutions, Knoxville, TN) at 3, 24, and 48 h post-injection (p.i.). The mice with A431/H9 tumor (range, 195 429 cubic mm for MORAb-009 and 239 700 cubic mm for B3) were injected iv with Zr-89-MORAb-009 (100 microCi/10 or 60 microg) or Zr-89-B3 (80 microCi/15 or 60 microg). All imaging procedures were performed under anesthesia with 1.5% isoflurane in oxygen at 2 L/min. The mice were then euthanized immediately after the 48 h imaging sessions for autoradiography. The PET images were reconstructed with a 3-dimensional ordered-subset expectation maximization/maximum a posteriori (OSEM3D/MAP) algorithm, with no attenuation or scatter correction. The reconstructed pixel size was 0.77 0.77 0.79 mm on a 128 128 159 imaging matrix. The image analysis was performed using ASIPro software (provided by Siemens, v6.8.0.0) on decay-corrected whole-body images. To characterize the accumulation of the probe in tumors, the region-of-interest (ROI) were drawn manually on individual tumor area, liver, spleen, muscle, and heart. The %ID/g was calculated for mice at 3, 24, and 48 h p.i.. For ex vivo autoradiography, the tumors were excised, embedded and frozen in Tissue-Tek CRYO-OCT compound (Sakura Finetek USA Inc., Torrance, CA, USA) at -80C for 3 h. Serial 16 micro-m thick short axis sections were cut in 400 micro-m intervals covering the entire tumor. Two or 3 tumor sections at 16 micro-m thickness were selected in 3 different tumor regions as representative sections throughout of the tumor and exposed in the phosphor screen for 16 h. Signals were obtained by the Typhoon FLA 7000 (GE Healthcare Life Sciences, Pittsburgh, PA, USA) with 25 micro-m pixel resolution and analyzed with Image Quant TL8.1 software. Values were grouped together from the 3 tumor regions to represent a tumor. Each tumor was treated as an independent sample (n=3). To analyze the microdistribution of the radioactivity in the tumor sections, a line was first drawn along a longest axis, and at the center of the longest line a second perpendicular line was drawn along a short axis. The center was selected as the point where two lines meet. Additional lines were drawn evenly and continuously between the two original lines passing through the same center point. Radioactivity profile of each line was analyzed with ImageJ and exported into Excel files to refine values with MATLABs interpolation function interp1. Results: The results of BD studies of Zr-89 MORAb-009 showed that the tumor uptake (2.72+/-0.63 for 2 microg vs. 6.13+/-2.27 %ID/g for 10 microg, p=0.03 and 12.33+/-2.55 %ID/g for 60 microg, p=0.02) and blood retention (1.94+/-0.42 for 2 microg vs. 3.71+/-3.43 %ID/g for 10 microg, p=0.32 and 9.48+/-1.95 %ID/g for 60 microg, p<0.001) increased as the injection dose was increased, whereas the liver uptake (31.68+/-7.47 for 2 microg vs. 25.34+/-4.56 %ID/g for 10 microg, p=0.15 and 19.59+/-3.83 %ID/g for 60 microg, p=0.02) gradually decreased as the injection dose was increased. In contrast, for Zr-89 B3, there was no appreciable dose effects shown on the tumor uptake (14.35+/-4.75 for 2 microg vs. 12.51+/-1.31 %ID/g for 15 microg, p=0.50 and 12.01+/-1.78 %ID/g for 60 microg, p=0.41), blood retention (16.78+/-1.03 for 2 microg vs. 15.80+/-1.49 %ID/g for 15 microg, p=0.27 and 16.29+/-0.96 %ID/g for 60 microg, p=0.46) and liver uptake (13.92+/-0.92 for 2 microg vs. 14.51+/-3.29 %ID/g for 15 microg, p=0.72 and 13.34+/-0.80 %ID/g for 60 microg, p=0.32) among the three different injection doses of Zr-89-B3. The findings from the BD studies were supported by the PET imaging studies as follows: The PET images visualized tumors as early as 3 h p.i. for both 10 and 60 microg MORAb-009 dose. At 24 and 48 h p.i., the radioactivity signal in the tumor remained relatively unchanged compared to that at 3 h p.i. for 10 microg whereas the tumor signal increased over time for 60 microg that is advantageous for the tumor visualization by PET. Compared to Zr-89 MORAb-009, the PET images from Zr-89 B3 did not show any significant dose effects on its uptake and the clearance pharmacokinetics from tumor, blood and liver. The PET images visualized tumors as early as 3 h p.i. for both 15 and 60 microg B3 dose. In addition, unlike Zr-89 MORAb-009, the radioactivity signal steadily increased over time for both B3 doses because Lewis-Y antigen is not shed from the tumor surface. The both BD and PET studies, thus, suggest that the shed antigen in the blood circulation negatively affects the antigen-specific tumor uptake of the mAb. To answer a second question if the shed antigen in the extracellular space of tumor could improve the penetration of mAb, we performed the autoradiography of tumor segments 2 days after the injection of Zr-89 MORAb-009 and Zr-89-B3. Our preliminary results indicate that for Zr-89-B3, higher radioactivity signals were consistently shown in the tumor periphery (>2 times), near the tumor surface than tumor core and diminished rapidly as moving toward the tumor core. In contrast, for Zr-89 MORAb-009, the distribution of the radioactivity was more uniform than that of Zr-64-B3. The high signal intensity in the periphery initially decreased and then increased again near the tumor core with the signal intensity in the tumor core comparable to that in the tumor periphery. Conclusion: The results of our studies provide a message that shed-antigen in the circulation negatively affects the antigen-specific tumor uptake of the antigen-specific antibody, whereas shed-antigen in the extracellular space positively affects the tumor uptake by improving the microdistribution of the antibody, which is important for antibody-based therapy.